RT-PCR - (Dec/17/2004 )

Hi !I am trying to amplify an inducible gene by using RT-PCR kit but either I am not getting the band or if I do slight changes in the conditions I just get the smear towards lower side. though I changed the primrrs but still I am having the problem. Could anyone suggst me something?

-promilasharma-

is your target gene only expressed when it's induced? if it is, i think you'd better find a cell line which constitutively expresses your interested gene. you can check your PRIMER or PCR efficiency with this cDNA. If both work very well, then proceed to your own cell to check its expression profile.

-littlecell-

Actually Iam doing in vivo study, not on the cell-line.I just induced the gene in an animal and then after induction isolated RNA from the tissue and then went for RT-PCR.

-promilasharma-

If you don't have the positive control, how can you know the failure of your RT-PCR resulted from the low quality of your RNA, the failure of RT, the failure of induction which means that there's no expression of your interested gene at all, or just PCR reaction? anyway, I think you should find the positive control.

-littlecell-

Thanks for your reply.One thing I want to know,is it possible to amplify a target gene of 4.9 kb completely using RT-PCR kit.